Phytochemistry 203 (2022) 113355

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Discovery of unreported ginkgolides of anti-PAF activity using

characteristic ion and neutral loss recognition strategy in Ginkgo biloba L
Jing Zhang 1, Jintang Cheng 1, Liu Yan, Yuetong Yu, Chenyang Hao, Anyi Zhao, Sha Chen *,
An Liu **
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences,
No. 16, Nanxiaojie, Dongzhimennei, Beijing, 100700, China

A R T I C L E I N F O A B S T R A C T

Keywords: Ginkgolides are the most important bioactive components of Ginkgo biloba L, of which ginkgolide B has been
Ginkgo biloba successfully developed and marketed as a drug. The reported ginkgolides are very rare and exhibit a complex
Ginkgoaceae matrix due to the chemodiversity of Ginkgo biloba L. Herein, the global profile of characteristic ion and neutral
Ginkgolide
loss recognition strategy were used for to discover eight undescribed ginkgolides, very rare cyclohexane ginkgolides
Characteristic ion
R–V, ginkgolides D–F, and eight known ginkgolides. These ginkgolides were target isolated and identified using
Neutral loss
Antiplatelet aggregation activities high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy, and X-ray crystallography. The
undescribed and known ginkgolides exhibited antiplatelet aggregation activities. In particular, compounds U and
D had IC50 values of 2.20 ± 0.15 and 6.50 ± 0.87 μM, respectively. This study has enriched the known structural
diversity of ginkgolides and extended the application of mass spectrometry to the global profiling of natural
products present in Ginkgo biloba L. Moreover, it could help chemists rapidly discover unreported compounds
from a complex matrix.

1. Introduction attention. Ginkgolides are a group of special Ginkgo biloba L. special

Ginkgo biloba L. (Ginkgoaceae), the last living representative of the
Ginkgoales, is called a “living fossil” because of its persistent morpho­
logical resemblance to its nearly 280-million-years-old fossil relatives.
Over the last century, Ginkgo biloba L. extracts have been used in dietary
supplements and herbal medicines for brain health (Chassagne et al.,
2019). Owing to its anti-ischemic, antioxidant, anticonvulsant, and
antiplatelet-activating factor activities as well as radical scavenging,
blood flow improvement, and blood vessel protection properties (Pietri
et al., 1997; Janssens et al., 1999; Akisü et al., 1998; Chung et al., 1999;
Ranchon et al., 1999; Chen et al., 1999; Lugasi and Horvahovich, 1999;
Sasaki and Hatta, 1999), Ginkgo biloba L. has attracted widespread
metabolites with distinctive structural features and significant biological
activities such as antiplatelet aggregation, anti-inflammatory activities,
nerve cell protection, memory repairing, and antitumor effects (Chen
et al., 2021; Czigle et al., 2018). Several ginkgolide compounds, such as
ginkgolide A, ginkgolide B, ginkgolide J and ginkgolide C, exhibit potent
antiplatelet aggregation activities (Stanislav et al., 2004; Ying et al.,
2020). Out of these, ginkgolide B has been successfully developed and
marketed as a new drug (Geng et al., 2018). However, there are mainly
11 ginkgolides (Fig. S1, Supporting Information), which have been
identified until now (Liu et al., 2022). Structural complexity and di­
versity of natural products present challenges in their synthesis and new
drug development. Untargeted separation may affect the discovery of

Abbreviations: DPI, Diagnostic product ion; EIC, Extracted ion chromatogram; HR-ESI-MS, High resolution- Electron spray ionization -Mass spectrometry; LC,
Liquid chromatography; LC/IT-TOF-MS, liquid chromatography ion-trap time-of-flight mass spectrometry; LC/QTOF-MS, liquid chromatography coupled with
quadrupole time-of-flight tandem mass spectrometry flight mass spectrometry; LC/IM-MS, liquid chromatography coupled with ion mobility quadrupole time-of-
flight mass spectrometry; NL, Neutral loss; QTOF-MS, Quadrupole time-of-flight mass spectrometry; TIC, Total ion chromatogram; UHPLC, Ultrahigh-perfor­
mance liquid chromatography; UHPLC-FT-MS, Ultrahigh-performance liquid chromatography coupled with Fourier transform mass spectrometry; UHPLC-MS-NMR,
Ultrahigh-performance liquid chromatography-mass spectrometry-nuclear magnetic resonance.
* Corresponding author.
** Corresponding author.
E-mail addresses: schen@icmm.ac.cn (S. Chen), aliu@icmm.ac.cn (A. Liu).
1
These authors contributed equally to the paper.

https://doi.org/10.1016/j.phytochem.2022.113355
Received 14 April 2022; Received in revised form 25 July 2022; Accepted 28 July 2022
Available online 7 August 2022
0031-9422/© 2022 Elsevier Ltd. All rights reserved.
J. Zhang et al.
ginkgolides (Beck and Stengel, 2016). The traditional ginkgolide sepa­
ration methods are time consuming and laborious, and it is challenging
to isolate ginkgolides from complex mixtures using conventional
repeated column chromatography (Meesakul et al., 2020; Wongsom­
boon et al., 2021). Therefore, efficient recognition and target-guided
isolation techniques are crucial to accelerate research on ginkgolides
for developing antiplatelet aggregation drugs.
Hyphenated analytical techniques are the major tools for discovering
novel natural products (Gaudencio and Pereira, 2015; Kleigrewe et al.,
2015). Different types of mass spectrometry techniques are hyphenated
with chromatography, including liquid chromatography ion-trap
time-of-flight mass spectrometry (LC/IT–TOF–MS), liquid chromatog­
raphy coupled with quadrupole time-of-flight tandem mass spectrom­
etry and flight mass spectrometry (LC/QTOF–MS),
ultrahigh-performance liquid chromatography coupled with Fourier
transform mass spectrometry (UHPLC–FT–MS), liquid chromatography
coupled with ion mobility quadrupole time-of-flight mass spectrometry
(LC/IM–QTOF–MS), and ultrahigh-performance liquid
chromatography-mass spectrometry-nuclear magnetic resonance
(UHPLC–MS–NMR), for allowing the rapid structural characterization of
unknown compounds from complex herbal extracts (Hao et al., 2008;
Yang et al., 2012; Hoffmann et al., 2014; Nielsen et al., 2011; Marchal
et al., 2015; Sun et al., 2011.; Schmidt et al., 2008.; Sprogøe et al., 2008).
However, these techniques are inefficient because they rely on the
precise spectral analysis (MS, MS/MS, and MSn) of each of the numerous
compounds, which is inappropriate for structurally complex natural
products. Furthermore, weak compound mass spectra may be missed
during data acquisition, yielding low identification resolution (Hao
et al., 2008; Yang et al., 2012). Structural cognition combined with the
detailed identification of the indigenous components of ginkgolides is
critical for the qualitative analysis of Ginkgo biloba L. and its products
(Yao et al., 2017, 2018; Qiao et al., 2016).
The workflow followed herein is presented in Fig. 1. First, we
developed an analysis strategy based on characteristic ion and neutral
loss scanning that can be used to globally profile compounds with
similar substructures. Second, the fractions passing through neutral
alumina chromatographic and silica gel columns were detected by
UHPLC-QTOF-HR-MS/MS, and the unreported ginkgolides are found by
Fig. 1. Flowchart showing global profiling and predicted metabolite screening
based on UHPLC–QTOF–HR–MS/MS-guided separation of ginkgolides from
Ginkgo biloba L.
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Phytochemistry 203 (2022) 113355
characteristic ion and neutral loss recognition. Then these fractions with
unreported ginkgolides are separated, and finally the undescribed
ginkgolides are targeted separation. Third, the unreported compounds
were identified using UHPLC–QTOF–HR–MS/MS, NMR spectroscopy,
and X-ray crystallography. Finally, the antiplatelet aggregation activ­
ities of the isolated ginkgolides were evaluated.
2. Results and discussion
2.1. Multiple characteristic ions and neutral loss analysis
The cleavage rules of the 11 known ginkgolides are summarized in
Fig. 2A. These rules are based on the six known reference substances of
ginkgolides (ginkgolide A, 31; ginkgolide B, 32; ginkgolide C, 33;
ginkgolide J, 34; bilobalide, 35; ginkgolide K, 36), which are used in the
UHPLC–QTOF–HR–MS/MS analysis; their fragmentation pathways are
shown in Figs. S2A–S2D, Supporting Information. The MS/MS spectra of
ginkgolides revealed significant characteristic ion of m/z 73. When
substituent R1 = OH and R3 = OH (Fig. 2A), the representative com­
pounds are 32, 33, and ginkgolide Q (38), exhibiting characteristic ions
of m/z 141 and m/z 125. When R1 = OH, C3=C16 has a double bond
(Fig. 2A), and the representative compounds are 36 and ginkgolide N
(41), with the characteristic ion of m/z 125. The compounds 31, gink­
golide M (39), ginkgolide P (37), 35, ginkgolide L (40), and 34 were
synthesized with no characteristic ion but were identified based on their
characteristic neutral losses of 56 and 74 Da.
The characteristic ion of m/z 73 is used as the substructure recog­
nition ion for ginkgolides in the multiple characteristic ions and neutral
losses strategy (Fig. 2B). The class I ginkgolides were identified by the
characteristic ion of m/z 125 or m/z 141. Based on the structure type, the
known ginkgolides are 32, 33, 36, 38, and 41, along with characteristic
fragments. Based on the structure type, the ions identified with neutral
losses of 56 and 74 Da are classified as class II ginkgolides, which include
31, 34, 35, 37, 39, and 40.
2.2. Data mining
Different fractions with characteristic and diagnostic ions were
deduced based on the UHPLC–QTOF–HR–MS/MS analysis for data
mining. These fractions were unambiguously identified after passing
through a neutral alumina chromatographic column as follows. First, a
target-extracted ion of m/z 73 was selected through total ion chroma­
tography (TIC). Second, the target compounds were identified as class I
ginkgolides based on an extracted ion chromatogram (EIC) image for
characteristic ions of m/z 73 combined with characteristic ions of m/z
141 or m/z 125. Conversely, characteristic ions of m/z 73 with neutral
losses of 56 or 74 Da were observed in the EIC image, and the com­
pounds were identified as class II ginkgolides. Overall, 25 ginkgolides
(11 known ginkgolides and 14 potentially previously unreported gink­
golides) were identified. The TIC and EIC images of the 14 potentially
undescribed ginkgolides are shown in Fig. 3A.
To obtain more ginkgolides of low content, silica gel was combined
with purified and enriched ginkgolides. For facilitating further investi­
gation, the fractions were analyzed after passing through a silica gel
chromatography column. First, the target-extracted ion of m/z 73 was
selected to enable the identification of various fractions. Second, the
target compounds were identified as class I ginkgolides based on the EIC
image of characteristic ions with m/z 73 combined with characteristic
ions of m/z 141 or m/z 125. Conversely, when ions with neutral losses of
56 or 74 Da were observed in the EIC image, the compounds were
identified as class II ginkgolides. In total, 16 previously unreported
ginkgolides were discovered and identified among the eight fractions,
their TIC and EIC fragmentation behaviors are shown in Fig. 3B.
Using this strategy, 41 ginkgolides were discovered in Ginkgo biloba
L., including 30 potentially undescribed compounds. These potential
unreported ginkgolides should be studied further.
J. Zhang et al.
Fig. 2. A: Cleavage rules of the known 11 ginkgolides. B: Diagram for identifying ginkgolides based on characteristic ion filtering, neutral loss, and substructure
recognition.
2.3. Structural identification of unreported ginkgolides and known
ginkgolides
Unreported ginkgolides (compounds 12, 13, 19, 21, 26, 6, 9, and
20), and eight known ginkgolides (compounds 31, 32, 33, 34, 35, 36,
37, and 38) were found and targeted for separation using a preparative
HPLC.
The structures of the undescribed ginkgolides together with the X-ray
crystallography patterns of compounds 12 and 6 are shown in Fig. 4. Key
HMBC, 1H–1H COSY, and NOESY correlations of the undescribed
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Phytochemistry 203 (2022) 113355
ginkgolides, i.e., compounds 12 (ginkgolide R), 13 (ginkgolide S), 19
(ginkgolide T), 21 (ginkgolide U), and 26 (ginkgolide V), 6 (Ginkgolide
D), 9 (Ginkgolide E), and 20 (Ginkgolide F) are shown in Fig. 5.
Compound 12, obtained as a colorless prism, was determined to have
the molecular formula of C20H28O9 based on High resolution- Electron
spray ionization -Mass spectrometry (HR-ESI-MS) (m/z 411.1663
[M–H]− , calculated m/z 411.1661 [M–H]− ). Its infrared (IR) spectrum
exhibited hydroxyl (3358–3552 cm− 1) and γ-lactone (1803 cm− 1)
functionalities. The 13C NMR spectra of compound 12 displayed 20
carbon signals, of which four signals were from methyl, three from
J. Zhang et al. Phytochemistry 203 (2022) 113355

Fig. 3. A: TIC and EIC of 14 potential unreported ginkgolides after passing through a neutral alumina chromatographic column. B: TICs and EICs of 16 potential
undescribed ginkgolides after passing through a silica gel chromatographic column.

methylene, six from methine (including one hemiacetal carbon), and
seven from quaternary carbon (including two carbonyl) groups. Based
on these results and the reported NMR data of ginkgolide derivatives, it
was suggested that compound 12 had the characteristic ginkgolide
skeleton structure. The X-ray crystallography pattern of GR is shown in
Fig. 4: C20H28O9, M = 412.42, crystal system = space group ortho­
rhombic, P2121, a = 10.331 (18) Å, b = 13.953 (3) Å, c = 15.967 (4) Å, V
= 2339.560 (7) Å3, Z = 4, and d = 1.224 g/cm3. A crystal with a size of
0.30 mm × 0.20 mm × 0.20 mm was used for measurements using a
Nonius CAD4/PC X-ray diffractometer, the θ value was set from 1.92◦ to
36.583◦ . The total number of independent reflections was 21,864, of
which 10,345 were observed (|F |2 ≥ 2σ| F |2). The crystal structure was
solved using the direct method SHELXS-97, and subsequently refined
using the program and SHELXS-97 and full-matrix least squares
methods. For the 21,864 observed reflections and 531 variable param­
eters, this refinement yielded a final Rf = 0.0725 and Rw = 0.2412. The
crystallographic data for the structure of 12 has been deposited at the
Cambridge Crystallographic Data Centre (deposition number CCDC
2156379).
Compound 13, obtained as a white colorless crystal, was determined
to have a molecular formula of C20H28O9 using HR-ESI-MS (m/z
411.1667 [M–H]− , calculated 411.1661 [M–H]− ). Its IR spectrum
showed absorptions at 3603 and 3359 cm− 1 corresponding to the hy­
droxyl group and 1780 cm− 1 corresponding to the carbonyl group. 13C

4
J. Zhang et al.
Fig. 4. Structures of eight unreported ginkgolides and X-ray crystallography of compound 12 and 6.
and DEPT–NMR spectra displayed 20 carbon signals. The 1H and 13C-
NMR data of compound 13 are similar to those of compound 12; how­
ever, a slight change in δC-14, δC-15, and δC-16 was identified. δC-14 moved
to a lower field, whereas δC-15 and δC-16 moved to a higher field. The
NOESY spectrum showed that H-2 is related to H-1 of compound 12 and
compound 13, whereas H-2 and H-14 of compound 12 are unrelated,
and H-2 is related to H-14 of compound 13. Therefore, it is speculated
that compound 13 is an isomer of compound 12, and isomerization
occurred at C-14. All hydrocarbons and their signals were accurately
determined using 2D-NMR (Table 1 and Fig. S4).
Compound 19, obtained as white colorless crystals, was determined
to have a molecular formula of C20H28O10 based on HR-ESI-MS (m/z
427.1615 [M-H]− , calculated m/z 427.1610 [M-H]− ). Its IR spectrum
showed absorptions at 3362–3554 and 1780 cm− 1 corresponding to the
hydroxyl and carbonyl groups, respectively. 1H and 13C-NMR data of
compound 19 showed an analogy to those of compound 12. In com­
parison to compound 12, compound 19 exhibited one additional hy­
droxyl group, and its δC-7 and δH-7 moved to a lower field. Additionally,
its HSQC spectra indicated that the number of H connected to C-7
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Phytochemistry 203 (2022) 113355
decreased. Therefore, it is speculated that a hydroxyl group is added to
C-7. In the NOESY spectrum, H-7 is observed to be related to H-8α,
suggesting that compound 19 is a derivative of compound 12 with a
hydroxyl group at the C-7 position. All hydrocarbons and their signals
were accurately determined using 2D-NMR (Table 1 and Fig. S5).
Compound 21, obtained as a white colorless crystal from MeOH, was
determined to have a molecular formula of C22H32O9 using HR-ESI-MS
(m/z 439.1978 [M–H]− , calculated m/z 439.1974 [M–H]− ). Its IR
spectrum showed absorptions at 3362–3554 and 1780 cm− 1 corre­
sponding to the hydroxyl and carbonyl groups, respectively. 13C and
DEPT-NMR spectra displayed 20 carbon signals. The 1H and 13C NMR
data of compound 21 were similar to those of compound 12. A com­
parison of 21 with 12 revealed that 21 has one more ethyl group, and the
δC-4 of 21 moved to a higher field. Thus, it is speculated that the ethyl
group is added to the hydroxyl group at the C-4 position such that 21
includes a hydroxyl group and ethyl group at the C-4 position of 12. All
hydrocarbons and their signals were accurately determined using 2D-
NMR (Table 1 and Fig. S6).
Compound 26, obtained as white colorless crystals, was determined
J. Zhang et al. Phytochemistry 203 (2022) 113355

Fig. 5. A: Key HMBC, 1H–1H COSY, and NOESY correlations of compound 12, 13, 19, 21, and 26. B: Key HMBC, 1H–1H COSY, and NOESY correlations of 6, 9,
and 20.

6
J. Zhang et al.
Fig. 5. (continued).
to have a molecular formula of C22H32O10 based on HR-ESI-MS (m/z
455.1926 [M-H]− , calculated m/z 455.1923 [M–H]− ). Its IR spectrum
showed absorptions at 3357–3663 and 1785 cm− 1 corresponding to the
hydroxyl and carbonyl groups, respectively. 13C and DEPT–NMR spectra
displayed 20 carbon signals. A comparison of compound 26 with com­
pound 19 revealed that 26 has one more ethyl group and the δC-4 of 26
moved to a higher field, indicating that the ethyl group is added to the
hydroxyl group at the C-4 position. Based on these results, 26 is
considered to contain hydroxyl and ethyl groups at the C-4 position of
19. All hydrocarbons and their signals were accurately determined using
2D-NMR (Table 1 and Fig. S7).
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Phytochemistry 203 (2022) 113355
Compound 6, obtained as white colorless crystals, Its IR spectra
revealed the presence of hydroxyl (3358–3552 cm− 1) and γ-lactone
(1802 cm− 1) functionalities. The molecular formula was determined as
C20H24O9 using HR-ESI-MS (m/z 407.1349 [M-H]− , calculated m/z
407.1345 [M–H]− ). 13C and DEPT–NMR spectra of compound 6 dis­
played 20 carbon signals, including four signals from methyl, two from
methylene, and six from methine (including one hemiacetal carbon)
groups as well as eight from quaternary carbons (including three car­
bonyls). From the above data, it was suggested that compound 6
possessed a characteristic ginkgolide skeleton.
When the 1H-NMR and 13C-NMR spectra of compound 6 are
J. Zhang et al. Phytochemistry 203 (2022) 113355

Table 1
1
H (500 MHz) and 13C NMR (125 MHz) data for compounds 12, 13, 19, 21, and 26 in methanol-d4, δ is in ppm, J is in Hz.
Proton Compound 12 Compound 13 Compound 19 Compound 21 Compound 26
δH δC δH δC δH δC δH δC δH δC
1α 2.16 (dd, J = 31.60 2.21 (dd, J = 31.62 1.99 (dd, J = 10.2, 26.17 2.01 (dd, J = 10.2, 12.6) 31.11 2.01 (dd, J = 10.2, 26.01
10.2, 12.6) 10.2, 12.6) 12.6) 12.6)
1β 2.81 (dd, J = 6.6, 2.93 (dd, J = 6.6, 2.89 (dd, J = 6.6, 2.85 (dd, J = 6.6, 12.6) 2.84 (dd, J = 6.6,
12.6) 12.6) 12.6) 12.6)
2 4.43 (dd, J = 6.6, 83.24 4.44 (dd, J = 6.6, 82.22 4.63 (dd, J = 6.6, 82.06 4.43 (dd, J = 6.6, 10.2) 82.57 4.62 (overlap) 80.72
10.2) 10.2) 10.2)
3 75.69 76.86 75.61 75.49 75.54
4α 77.38 77.51 77.92 71.42 71.42
4β 3.62 (s) 3.71 (s) 3.67 (s) 3.85 (overlap) 3.88 (s)
5 110.27 110.27 110.41 112.37 112.91
6 58.94 59.01 62.94 60.46 64.62
7α 2.43 (m) 31.17 2.44 (m) 31.62 4.47 (m) 71.31 2.60 (m) 30.33 4.58 (overlap) 71.11
7β 1.61 (m) 1.63 (m) 1.57 (m)
8α 2.01 (m) 27.65 2.03 (m) 27.83 2.48 (overlap) 39.56 1.97 (m) 27.55 2.45 (m) 40.01
8β 1.93 (m) 1.91 (m) 1.79 (m) 1.88 (m) 1.72 (m)
9 2.34 (dd, J = 4.2, 51.80 2.35 (dd, J = 4.2, 51.53 2.47 (overlap) 49.65 2.34 (dd, J = 4.2, 8.4) 51.40 2.28 (t, J = 10.5) 50.15
8.4) 8.4)
10 66.44 66.62 66.37 66.28 66.24
11 4.99 (s) 69.89 4.98 (s) 70.1 5.00 (s) 69.11 4.97 (s) 69.54 4.99(s) 68.96
12 176.13 176.08 175.22 175.92 174.93
13 5.98 (s) 105.91 6.0 (1H, s) 105.96 5.95 (s) 106.18 6.0 (1H, s) 106.40 5.92(s) 106.49
14 2.99 (q, J = 7.8) 41.96 2.77 (q, J = 6.6) 45.58 2.96 (q, J = 7.8) 42.32 2.96 (1H, q, 8) 42.49 2.91 (q, J = 7.8) 43.12
15 180.23 177.96 180.23 180.49 180.49
16 1.24 (d, J = 7.8) 9.93 1.32 (d, J = 6.6) 6.41 1.24 (d, J = 7.8) 10.41 1.24 (1H, overlap) 10.56 1.24 (d, J = 7.8) 10.96
17 32.43 32.43 32.53 32.47 32.62
18 1.09 (s) 28.43 1.09 (s) 28.43 1.07 (s) 28.13 1.09 (s) 28.10 1.07 (s) 28.10
19 1.09 (s) 28.43 1.09 (s) 28.43 1.07 (s) 28.13 1.09 (s) 28.10 1.07 (s) 28.10
20 1.09 (s) 28.43 1.09 (s) 28.43 1.07 (s) 28.13 1.09 (s) 28.10 1.07 (s) 28.10
21 3.79 (dd, J = 7, 16), 3.68 57.00 3.76 (dd, J = 7, 56.94
(dd, J = 7, 16) 16),
3.68 (dd, J = 7,
16)
22 1.22 (overlap) 14.86 1.20 (t, J = 7) 14.57

compared with compound 31 (Table 2), δC-2, δC-3, δC-4, and δC-5 shifted
to lower magnetic fields. The δH-1 and δH-2 also shifted: δH-1 shifted to the
lower magnetic field, whereas δH-2 shifted to the higher field. Moreover,
the coupling between H-1 and H-2 changed; in 31, H-2 coupled with H-
1α and H-1β, splitting into a triplet, whereas in 6, H-2 only coupled with
H-1α, splitting into a doublet. X-ray crystallography patterns of 6
showed that 6 is an isomer of 31 and that isomerism occurred at the C-14
position. All hydrocarbons and their signals were accurately determined
using 2D-NMR and single crystal diffraction technology (Table 2, Fig. 4,
and Fig. S8).
The X-ray crystallography pattern of compound 6 is shown in Fig. 4:
C20H24O9, M = 440.43, crystal system = space group orthorhombic,
P212121, a = 10.60000 (18) Å, b = 11.80600 (3) Å, c = 18.69500 (4) Å,
V = 2339. 560 (7) Å3, Z = 4, d = 1.250 g/cm3. A crystal with size of 0.30
mm × 0.20 mm × 0.20 mm was used for measurements using a Nonius
CAD4/PC X-ray diffractometer, the θ value was set from 1.74◦ to 25.04◦ .
The total number of independent reflections measured was 4093, of
which 2356 were observed (|F |2 ≥ 2σ| F |2). The crystal structure was

Table 2
1
H (500 MHz) and 13C NMR (125 MHz) data for compounds 31, 6, 33, and 9 in methanol-d4, δ in ppm, J in Hz.
Proton Compound 31 Compound 6 Compound 33 Compound 9
δH δC δH δC δH δC δH δC
1α 1.88 (dd, J = 5, J = 14) 36.56 2.73 (dd, J = 18, J = 6) 36.86 73.85 75.55
1β 2.78 (dd, J = 7.5, J = 15.5) 3.21 (d, J = 18) 4.13 (d, J = 7) 4.97 (s)
2 4.77 (t, J = 7.5) 88.31 4.66 (d, J = 6) 89.94 4.55 (d, J = 7) 91.93 4.45 (s) 95.16
3 86.48 88.01 83.21 86.96
4 100.94 104.81 98.49 104.42
5 68.7 72.32 66.73 70.89
6 4.85 (overlap) 86.4 4.81 (d, J = 4) 88.57 5.10 (d, J = 4) 79.34 5.28 (d, J = 4.2) 79.76
7α 2.14 (m) 36 2.13 (m) 36.16 4.21 (m) 74.79 4.34 (dd) 74.31
7β 2.2 (m) 2.24 (m)
8 2.07 (m) 49.14 1.90 (m) 49.81 1.76 (d, J = 12.5) 49.66 1.78 (d, J = 12) 50.1
9 67.43 67.31 64.1 64.18
10 5.02 (s) 68.97 5.01 (s) 69.35 5.10 (d, J = 5.0) 69.15 5.05 (s) 69.08
11 174.4 173.9 173.63 173.92
12 6.02 (s) 110.24 5.94 (s) 108.76 6.09 (s) 110.34 5.94 (s) 108.46
13 171.91 172.66 171.09 172.7
14 3.12 (q, J = 7) 40.81 2.97 (q, J = 9.8) 44.34 2.98 (q, J = 7) 41.89 2.94 (q, J = 9.8) 44.44
15 177.44 175.55 176.89 175
16 1.23 (d, J = 7.5) 7.19 1.45 (d, J = 10.5) 7.01 1.22 (d, J = 7) 6.67 1.43 (d, J = 9.8) 6.91
17 31.89 31.94 31.88 32.01
18 1.11 (s) 28.12 1.11 (s) 28.12 1.19 (s) 28.12 1.21 (s) 28.2
19 1.11(s) 28.12 1.11(s) 28.12 1.19 (s) 28.12 1.21(s) 28.2
20 1.11 (s) 28.12 1.11 (s) 28.12 1.19 (s) 28.12 1.21 (s) 28.2

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J. Zhang et al. Phytochemistry 203 (2022) 113355

solved using the direct method SHELXS-97, and subsequently refined 2004).
using the program and methods SHELXS-97 and full-matrix least
-squares. For the 4093 observed reflections and 286 variable parame­
ters, the refinement yielded Rf = 0.0723 and Rw = 0.2219. The crys­ 2.4. Antiplatelet aggregation activity assay
tallographic data for the structure of compound 1 has been deposited at
the Cambridge Crystallographic Data Centre (deposition number CCDC Previous studies demonstrated that the ginkgolides have inhibitory
2117254). activities against platelet aggregation induced by the platelet-activating
Compound 9, obtained as white colorless crystals, was determined to factor (PAF). compound 31, 32, 33, 12, 13, 19, 21, 26, 6, 9, and 20
have a molecular formula of C20H24O11 using HR-ESI-MS (m/z 439.1249 (purity >95%) extracted from the leaves of Ginkgo biloba L. were eval­
[M-H]− , calculated m/z 439.1246 [M–H]− ). The IR spectrum showed uated for inhibitory activities against rabbit platelet aggregation using
absorptions at 3603 and 3359 cm− 1 corresponding to the hydroxyl compound 32 as a positive control. The results demonstrated that
group and 1780 cm− 1 assigned to the carbonyl group. Comparing the compound 31, 33, 32, 12, 13, 19, 21, 26, 6, 9, and 20 have inhibitory
1
H-NMR and 13C-NMR spectra of 9 with 33 (Table 2), the δC-2, δC-3, δC-4, activities with IC50 values of 4.61 ± 0.65, 13.2 ± 0.58, 0.61 ± 0.23,
and δC-5 shifted to lower magnetic fields. δH-1 and δH-2 also shifted: δH-1 19.34 ± 0.68, 13.83 ± 0.92, 23.20 ± 1.24, 32.45 ± 2.12, 46.52 ± 2.78,
moved to the lower magnetic field, whereas δH-2 moved to the higher 6.50 ± 0.87, 16.32 ± 0.64, and 2.20 ± 0.15 μM, respectively. Table 4
field. Moreover, H-2 was coupled with H-1β in 33, splitting into two summarizes the activities of ginkgolides on PAF in vitro. Based on the
peaks; however, H-2 exhibited only a single peak in GE. These differ­ activities of the known 31, 33, and 32 with those of compound 6, 9, and
ences are the same as those between 6 and 31. The NOESY spectrum of 9 20, it is hypothesized that isomerization at the C-14 position slightly
showed that H-14 is related to H-2, therefore, it is deduced that 9 is an decreased the activity of antiplatelet aggregation induced by PAF. When
isomer of 31 and isomerism occurred at C-14 position. All hydrocarbons comparing the activities of compound 12 and 13, it is suggested that an
and their signals were accurately determined using 2D-NMR and are outward methyl group is present at the C-14 position, which reduces the
shown in Table 2 and Fig. S9. activity of antiplatelet aggregation induced by PAF. Based on the ac­
Compound 20: obtained as white colorless crystals, the molecular tivities of compound 12 and 19, the hydroxyl group is observed to be
formula was determined as C20H24O10 using HR-ESI-MS (m/z 423.1295 connected at the C-7 position, decrease the activity of antiplatelet ag­
[M–H]− , calculated m/z 423.1297 [M–H]− ). The IR spectrum showed gregation induced by PAF. Finally, based on the activities of compound
absorptions at 3603 and 3359 cm− 1 ascribed to the hydroxyl group and 21 and 26, the hydroxyl group is observed to be connected at the C-7
1780 cm− 1 ascribed to the carbonyl group. 1H-NMR and 13C-NMR position, reducing the activity of antiplatelet aggregation induced by
spectra of 20 are compared with 32 (Table 3), which indicated that δC-2, PAF.
δC-3, δC-4, and δC-5 shifted to lower magnetic fields. δH-1 moved to the
lower magnetic field and δH-2 moved to the higher magnetic field, 3. Conclusions
indicating that the coupling between H-1 and H-2 changed in 32.
Further, in 32, H-2 is coupled with H-1β, which shows splitting into a Herein, unreported ginkgolides, i.e., compound 12, 13, 19, 21, 26, 6,
double peak, whereas in 20, H-2 is not coupled with H-1β, which showed 9, and 20, and eight known ginkgolides were targeted, separated, pu­
a single peak. These changes are the same as those observed between 31 rified, and identified using UHPLC–QTOF–HR–MS/MS, NMR spectros­
and 6. The NOESY spectrum of 20 indicated that H-14 is related to H-2. copy, and X-ray diffraction. These ginkgolides supplied complex and
Based on these results, it could be speculated that 20 is an isomer of 32, diverse natural products and served as important compounds for
with the isomerism occurring at the C-14 position. All hydrocarbons and developing new drugs. These compounds were determined using mul­
their signals were accurately determined using 2D-NMR and are shown tiple characteristic ions and neutral loss ions combined with substruc­
in Table 3 and Fig. S10. ture recognition. In addition, the antiplatelet aggregation activities of
The known ginkgolides, compound 31, 32, 33, 34, 35, 36, 37, and the novel ginkgolides indicated that multiple components work together
38 were identified by related literature (Liao et al., 2011; Lou et al., in the clinical effects of this herbal medicine and the observed pre­
liminary relationships between the structure and activity are helpful for
developing new drugs. In summary, this study has opened a new
Table 3
1
H (500 MHz) and 13C NMR (125 MHz) data for compounds 32 and 20 in
Table 4
methanol-d4, δ in ppm, J in Hz.
Activity of ginkgolides on PAF in vitro (±s, n = 3).
proton Compound 32 Compound 20
Group Drug concentration Platelet aggregation IC50
δH δC δH δC
(μmol L− 1) inhibition rate (%) (μmol⋅L− 1)
1α 74.05 75.73
1β 4.18 (d, J = 10) 5.00 (s) Compound 10 83.26 ± 3.21 4.61 ± 0.65
2 4.56 (d, J = 10) 91.9 4.46 (s) 95.40 31
3 83.24 87.00 Compound 10 48.17 ± 2.32 13.2 ± 0.58
4 98.78 104.94 33
5 72.11 76.59 Compound 10 97.21 ± 0.32 0.61 ± 0.23
6 5.39 (d, J = 4.5) 79.3 5.50 (brs) 79.38 32
7α 2.06 (m) 36.74 2.12 (m) 36.20 Compound 10 30.96 ± 0.45 19.34 ±
7β 2.24 (m) 2.10 (m) 12 0.68
8 1.88 (m) 49.06 1.93 (m) 49.96 Compound 10 43.23 ± 1.98 13.83 ±
9 67.78 67.95 13 0.92
10 5.01 (s) 69.26 5.01 (s) 69.36 Compound 10 28.48 ± 0.63 23.20 ±
11 173.79 173.99 19 1.24
12 6.07 (s) 110.49 5.92 (s) 108.54 Compound 10 24.61 ± 0.67 32.45 ±
13 171.25 172.90 21 2.12
14 3.0 (q, J = 7) 41.91 2.95 (q, J = 7) 44.63 Compound 10 20.64 ± 0.58 46.52 ±
15 176.96 175.03 26 2.78
16 1.21 (d, J = 7) 6.65 1.43 (d, J = 7) 6.84 Compound 6 10 64.50 ± 0.58 6.50 ± 0.87
17 31.91 31.93 Compound 9 10 31.30 ± 0.58 16.32 ±
18 1.12 (s) 28.09 1.11 (s) 28.24 0.64
19 1.12 (s) 28.09 1.11 (s) 28.24 Compound 10 89.56 ± 0.58 2.20 ± 0.15
20 1.12 (s) 28.09 1.11 (s) 28.24 20

9
J. Zhang et al.
window for ginkgolides as potential candidates for discovering and
developing new drugs as well as for discovering unknown functional
molecules in herbal medicines to support its clinical use. This study has
extended our understanding of ginkgolides in Ginkgo biloba L. Further­
more, multiple characteristic ions and neutral loss ions combined with
substructure recognition could be applied for rapidly and efficiently
discovering new bioactive constituents in other herbal medicines.
4. Experimental
4.1. General experimental procedures
IR spectra were acquired using a Nicolet 5700 FT-IR microscope
spectrometer (FT-IR microscope transmission, Thermo Electron Corpo­
ration, Madison, WI, USA). 1D and 2D-NMR spectra were obtained at
500 MHz for 1H and at 150 MHz for 13C, respectively, using a SYS 600
MHz spectrometer (Varian Associates Inc., Palo Alto, CA, USA) using
either the residual peak of HDO (δH = 4.800) or the methyl peak of
MeOH (δC = 49.500) as references. HR–ESI–MS data were acquired
using an Agilent 6540 Q-TOF mass spectrometer (Agilent Technologies,
Ltd., Santa Clara, CA, USA). Isolation was monitored using an Agilent
1100 HPLC (Agilent Technologies, Ltd., Santa Clara, CA, USA) equipped
with an Alltech 2000 ELSD detector (Alltech Technologies, Los Angeles,
USA).
4.2. Chemicals and reagents
Pure distilled water was purchased from Watson’s water (Hong
Kong, China). MS-grade methanol was purchased from Fisher Scientific
(Fair Lawn, NJ, USA). MS-grade formic acid was purchased from Sigma-
Aldrich (Sigma-Aldrich, MO, USA). Reference standards, compound 35
(>98%), 34 (>98%), 33 (>98%), 31 (>98%), 32 (>98%), and 36
(>98%), were purchased from Chengdu Must Biotechnology (Chengdu,
China).
4.3. Plant materials
Leaves of Ginkgo biloba L. (Ginkgoaceae) were bought from the
medicinal material market of Bozhou, Anhui Province, P.R. China (GPS
data; North: 33.844582, East: 115.778676) in October 2020. Dr. Wei
Sun, a taxonomist, identified the leaves as Ginkgo biloba L. (Institute of
Chinese Materia Medica, China Academy of Chinese Medical Sciences,
Beijing, China). A voucher specimen 202010 M was deposited in the
herbarium of the Department of Medicinal Plants, Institute of Chinese
Materia Medica, China Academy of Chinese Medical Sciences (Beijing,
China).
4.4. UHPLC–QTOF-MS/MS analysis conditions
Chromatographic separation was achieved using an ACQUITY
UPLC®BEH C18 (100 mm × 2.1 mm, 1.7 μm), with a column tempera­
ture of 40 ◦ C, autosampler temperature of 4 ◦ C, and injection volume
was of 2 μL. The mobile phase (at 0.35 mL/min) comprised 0.1% formic
acid in water (A) and methanol (B). The following were the elution
conditions: 0–2 min, 5%–10% B; 2–10 min, 10%–14% B; 10–13 min,
14%–14% B (run for 3 min with the same gradient in 14% to separate
these compounds); 13–29 min, 14%–30% B; 29–37 min, 30%–60% B;
37–45 min, 60%–95% B; 45–47 min, 95%–100% B; and 47–50 min,
95%–100% B.
An Agilent 6540 Q-TOF mass spectrometer equipped with a dual ESI
electrospray ion source was used in negative modes. Mass ranges were
set at m/z 50–1250 Da for the TOF–MS/MS experiments. For the
IDA–MS/MS experiment, the collision energy was set at 40 eV. The
collision energy spread was set at 10 eV (±) for the
UHPLC–QTOFMS–MS detection experiment. The ESI source operation
parameters and ESI–MS conditions were as follows: 325 ◦ C, 5-L/min gas
10
Phytochemistry 203 (2022) 113355
flow, 35-psi nebulizer pressure, and sheath gas temperature of 250 ◦ C.
To modify the measured masses in real time, internal references (purine
and HP-0921) were used, and the reference masses in the negative-ion
mode were m/z 119.0363 and m/z 1033.9881. LC–MS/MS data were
analyzed using qualitative analysis B.07.00.
4.5. Extraction and isolation
Pulverized leaves of Ginkgo biloba L. (10.0 kg) were extracted by
refluxing with 90% ethanol (100 L × 2). The combined extracts were
concentrated in vacuo to obtain the residue (1042 g). Finally, 2 mg/mL
of the residue solution was purified before the UHPLC–QTOF–MS/MS
analysis. The residues purified through the neutral alumina chroma­
tography column were dissolved in an ethanol–water solution (1500
mL:5000 mL, v:v). Subsequently, the filtrate was concentrated to 3000
mL and stewed at 4 ◦ C for 12 h. The residue (112 g) was subjected to a
silica gel chromatographic column (1.5 g, 10 cm × 100 cm) with etha­
nol–dichloromethane (1:120–1:0 ethanol, 6 L each) as an eluent to yield
10 fractions (Fr. 1–10). Next, 2 mg/mL of the eight fractions were
filtered before the UHPLC–QTOF–MS/MS analysis. Using characteristic
ion and neutral loss recognition strategy to find known and unreported
ginkgolides. First, connecting the YMC-Pack ODS-A (5 m, 250 × 4.6 mm,
Japan) to the UHPLC–QTOF–MS/MS to find suitable liquid chroma­
tography conditions to separate the ginkgolides. Second, in the prepar­
ative liquid chromatography, using the same column and liquid
chromatography conditions, repeat the UHPLC–QTOF–MS/MS results,
and then target separation of ginkgolides.
Fr. 2–6 were chromatographed on the YMC-Pack ODS-A using
CH3OH–H2O with 0.1% formic acid (2.5 mL/min). Fr. 2 (101.25 mg)
was purified with an isocratic elution (30% CH3OH for 45 min) to yield
compound 6 (23.47 mg), compound 9 (25.34 mg), compound 12 (13.21
mg), and compound 13 (3.15 mg). Fr. 3 (110.25 mg) was purified with
an isocratic elution (40% CH3OH for 40 min). Finally, compound 19
(5.20 mg), compound 20 (23.36 mg), and compound 21 (6.14 mg) were
obtained. Fr. 4 (12.12 mg) was purified with an isocratic elution (45%
CH3OH for 40 min), yielding compound 26 (3.35 mg). Fr. 5 (500.18 mg)
was purified with an isocratic elution (40% CH3OH for 30 min) to yield
compound 31 (55.24 mg), compound 32 (83.27 mg), compound 33
(55.16 mg), and compound 34 (45.27 mg). Fr. 6 (300.23 mg) was pu­
rified with an isocratic elution (30% CH3OH for 30 min) to obtain
compound 35 (22.37 mg), compound 36 (42.24 mg), compound 37
(15.23 mg), and compound 38 (22.45 mg).
Compound 6: White colorless crystals. mp > 300 ◦ C. IR (KBr) νmax:
3358–3552, 2996, 2960, 2917, 2875, 1802, 1745, 1647, 1457, 1374,
1355, 1335, 1272, 1227, 1135, 1098, and 1047 cm− 1. HR–ESI–MS m/z
407.1349 [M–H]− , calculated for C20H24O9 [M–H]− , m/z 407.1345.
Compound 9: White colorless crystals. mp > 300 ◦ C. IR (KBr) νmax:
3356–3550, 2995, 2964, 2915, 2873, 1805, 1747, 1646, 1457, 1378,
1358, 1337, 1273, 1228, 1135, 1094, and 1048 cm− 1. HR–ESI–MS m/z
439.1249 [M–H]− , calculated for C20H24O11 [M–H]− , m/z 439.1246.
Compound 12: White colorless crystals. mp > 300 ◦ C. IR (KBr) νmax:
3358–3552, 2996, 2960, 2917, 2875, 1802, 1745, 1647, 1457, 1374,
1355, 1335, 1272, 1227, 1135, 1098, and 1047 cm− 1. HR–ESI–MS m/z
411.1663 [M–H]− , calculated for C20H28O9 [M–H]− , m/z 411.1661.
Compound 13: White colorless crystals. mp > 300 ◦ C. IR (KBr) νmax:
3359–3603, 2994, 2962, 2915, 2877, 1801, 1747, 1644, 1455, 1378,
1357, 1332, 1276, 1221, 1137, 1092, and 1039 cm− 1. HR–ESI–MS m/z
411.1667 [M–H]− , calculated for C20H28O9 [M–H]− , m/z 411.1661.
Compound 19: White colorless crystals. mp > 300 ◦ C. IR (KBr) νmax:
3362–3554, 2998, 2962, 2919, 2873, 1780, 1745, 1643, 1457, 1376,
1356, 1332, 1274, 1229, 1136, 1093, and 1043 cm− 1. HR–ESI–MS m/z
427.1615 [M–H]− , calculated for C20H28O10 [M–H]− , m/z 427.1610.
Compound 20: White colorless crystals. mp > 300 ◦ C. IR (KBr) νmax:
3354–3552, 2995, 2966, 2917, 2875, 1805, 1747, 1648, 1458, 1376,
1358, 1338, 1336, 1276, 1229, 1136, 1094, and 1048 cm− 1. HR–ESI–MS
m/z 423.1295 [M–H]− , calculated for C20H24O10 [M–H]− , m/z
J. Zhang et al.
423.1297.
Compound 21: White colorless crystals. mp > 300 ◦ C. IR (KBr) νmax:
3362–3554, 2998, 2964, 2915, 2873, 1802, 1780, 1646, 1454, 1378,
1352, 1331, 1274, 1225, 1134, 1097, and 1045 cm− 1. HR–ESI–MS m/z
439.1978 [M–H]− , calculated for C22H32O9 [M–H]− , m/z 439.1974.
Compound 26: White colorless crystals. mp > 300 ◦ C. IR (KBr) νmax:
3357–3663, 2994, 2962, 2917, 2873, 1801, 1780, 1645, 1453, 1376,
1358, 1336, 1270, 1223, 1132, 10986, and 1049 cm− 1. HR–ESI–MS m/z
455.1926 [M–H]− , calculated for C22H32O10 [M–H]− , m/z 455.1923.
4.6. Antiplatelet aggregation activity assay
Antiplatelet aggregation activities of compounds 6, 9, 12, 13, 19, 20,
21, 26, 31, 32, and 33 were determined by PAF. PAF activities were
determined on isolated platelets prepared from New Zealand white
rabbits weighing 2.5–3.5 kg (Nanjing, China) following a previously
reported method (Li et al., 2002). Compound 32 (National Institute for
the Control of the Pharmaceutical and Biological Products, purity
>98%) was used as the positive control.
Funding
This work was supported by the Scientific and Technological Inno­
vation Project of China Academy of Chinese Medical Sciences [grant
numbers CI2021A4404 and CI2021A04405], the Major Scientific and
Technological Special Project for “the Fundamental Research Funds for
the Central public welfare research institutes” (Nos. ZZ13-YQ-057,
ZZ13- YQ-061 and ZXKT21006), and the National Key Research and
Development Project [grant number 2019YFC19066-01]. The funders
had no role in the study design, data collection, and analysis, decision to
publish, or preparation of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
An Liu and Sha Chen designed and guided all the experiments,
checked the data, and revised the manuscript. Jing Zhang and Jintang
Cheng conducted the chemical experiments, analyzed the data, and
wrote the preliminary manuscript. Yan Liu provided assistance with
language and writing. Yuetong Yu, Chenyang Hao, and Anyi Zhao proof
read the article. All the authors read and approved the final manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.phytochem.2022.113355.
References
Akisü, M., Kültürsay, N., Coker, I., Hüseyinov, A., 1998. Platelet-activating factor is an
important mediator in hypoxic ischemic brain injury in the newborn rat. Flunarizine
and Ginkgo biloba extract reduce PAF concentration in the brain. Biol. Neonate 74,
439–444. https://doi.org/10.1159/000014065.
Beck, S., Stengel, J., 2016. Mass spectrometric imaging of flavonoid glycosides and
biflavonoids in Ginkgo biloba L. Phytochemistry 130, 201–206. https://doi.org/
10.19540/j.cnki.cjcmm.20180312.001.
Chassagne, F., Huang, X., Lyles, J.T., Quave, C.L., 2019. Validation of a 16th century
traditional Chinese medicine use of ginkgo biloba as a topical antimicrobial. Front.
Microbiol. 10, 775. https://doi.org/10.3389/fmicb.2019.00775.
11
Phytochemistry 203 (2022) 113355
Chen, C., Wei, T.T., Gao, Z., Zhao, B., Hou, J., Xu, H., Xin, W., Packer, L., 1999. Different
effects of the constituents of EGb761 on apoptosis in rat cerebellar granule cells
induced by hydroxyl radicals. Biochem. Mol. Biol. Int. 47, 397–405. https://doi.org/
10.1016/s0300-483x(00)00200-6.
Chen, X.X., Zhong, W., Shu, C.Q., Yang, H., Li, E.H., 2021. Comparative analysis of
chemical constituents and bioactivities of the extracts from leaves, seed coats and
embryoids of Ginkgo biloba L. Nat. Prod. Res. 23, 5498–5501. https://doi.org/
10.1080/14786419.2020.1788020.
Chung, H.S., Harris, A.J., Kristinsson, J.K., Ciulla, T.A., Kagemann, C., Ritch, R., 1999.
Ginkgo biloba extract increases ocular blood flow velocity. J. Ocul. Pharmacol.
Therapeut. 15, 233–240. https://doi.org/10.1089/jop.1999.15.233.
Czigle, S., Toth, J., Jedlinszki, N., Haznagy-Radnai, E., Csupor, D., Tekel’ova, D., 2018.
Ginkgo biloba food supplements on the european market - adulteration patterns
revealed by quality control of selected samples. Planta Med. 84, 475–482. https://
doi.org/10.1055/a-0581-5203.
Gaudencio, S., Pereira, P.F., 2015. Dereplication: racing to speed up the natural products
discovery process. Nat. Prod. Rep. 32, 779–810. https://doi.org/10.1039/
c4np00134f.
Geng, T., Shen, W.W., Wang, J.J., Huang, W.Z., Wang, Z.Z., Xiao, W., 2018. Research
development of ginkgo terpene lactones. Zhongguo Zhongyao Zazhi 43, 1384–1391.
Hao, H., Cui, N., Wang, G., Xiang, B., Liang, Y., Xu, X., Zhang, H., Yang, J., Zhang, C.,
Wu, L., Gong, P., Wang, W., 2008. Global detection and identification of nontarget
components from herbal preparations by liquid chromatography hybrid ion trap
time-of-flight mass spectrometry and a strategy. Anal. Chem. 80, 8187–8194.
https://doi.org/10.1021/ac801356s.
Hoffmann, T., Krug, D., Hüttel, S., Müller, R., 2014. Improving natural products
identification through targeted LC-MS/MS in an untargeted secondary metabolomics
workflow. Anal. Chem. 86, 10780–10788. https://doi.org/10.1021/ac502805w.
Janssens, D., Remacle, J., Drieu, K., Michiels, C., 1999. Protection of mitochondrial
respiration activity by bilobalide. Biochem. Pharmacol. 58, 109–119. https://doi.
org/10.1016/s0006-2952(99)00061-1.
Kleigrewe, K., Almaliti, J., Tian, I.Y., Kinnel, R., Korobeynikov, B.A., Monroe, E.A.,
Duggan, B.M., Marzo, V.D., Sherman, D.H., Dorrestein, P.C., Gerwick, L.,
Gerwick, W.H., 2015. Combining mass spectrometric metabolic profiling with
genomic analysis: a powerful approach for discovering natural products from
cyanobacteria. J. Nat. Prod. 78, 1671–1682. https://doi.org/10.1021/acs.
jnatprod.5b00301.
Li, L., Li, M., Shen, Y.M., Chen, Z.H., Du, X.B., Hao, X., 2002. Antipatelet aggregation
activities of diterpene alkaloids from Spiraea fritschiana var. parvifolia. J. Nat. Prod.
Res. Dev. 14, 7–10. https://doi.org/10.1002/mop.10502.
Liao, H.J., Zheng, Y.F., Li, H.Y., Guo, P.P., 2011. Two new ginkgolides from the leaves of
Ginkgo biloba. Planta Med. 77, 1818–1821. https://doi.org/10.1055/s-0030-
1271153.
Liu, X.G., Lu, X., Gao, W., Li, P., Yang, H., 2022. Structure, synthesis, biosynthesis, and
activity of the characteristic compounds from Ginkgo biloba L. Nat. Prod. Rep. 39,
474–511. https://doi.org/10.1039/d1np00026h.
Lou, F.C., Lin, Y., Tang, Y.P., Wang, Y., 2004. Isolation, purification and structure
identification of ginkgo terpene lactones. Chin. J. Nat. Med. 1, 12–16.
Lugasi, A., Horvahovich, P., 1999. Additional information to the in vitro antioxidant
activity of Ginkgo biloba L. Phytother Res. 13, 160–162.
Marchal, A., Génin, E., Waffo-Téguo, P., Bibès, A., Da Costa, G., Mérillon, J.M.,
Dubourdieu, D., 2015. Development of an analytical methodology using Fourier
transform mass spectrometry to discover new structural analogs of wine natural
sweeteners. Anal. Chim. Acta 853, 425–434. https://doi.org/10.1016/j.
aca.2014.10.039.
Meesakul, P., Jaidee, W., Richardson, C., Andersen, R.J., Patrick, B.O., Willis, A.C.,
Muanprasat, C., Wang, J., Lei, X., Hadsadee, S., Jungsuttiwong, S., Pyne, S.G.,
Laphookhieo, S., 2020. Styryllactones from Goniothalamus tamirensis.
Phytochemistry 171, 112248. https://doi.org/10.1016/j.phytochem.2019.112248.
Nielsen, K.F., Mansson, M., Rank, C., Frisvad, J.C., Larsen, T.O., 2011. Dereplication of
microbial natural products by LC-DAD-TOFMS. J. Nat. Prod. 74, 2338–2348.
https://doi.org/10.1021/np200254t.
Pietri, S., Maurelli, E., Drieu, K., Culcasi, M., 1997. Cardioprotective and anti-oxidant
effects of the terpenoid constituents of Ginkgo biloba extract (EGb 761). J. Mol. Cell.
Cardiol. 29, 733–742. https://doi.org/10.1006/jmcc.1996.0316.
Qiao, X., Lin, X.H., Ji, S., Zhang, Z.X., Bo, T., Guo, D.A., Ye, M., 2016. Global profiling
and novel structure discovery using multiple neutral loss/precursor ion scanning
combined with substructure recognition and statistical analysis (MNPSS):
characterization of terpene-conjugated curcuminoids in curcuma longa as a case
study. Anal. Chem. 88, 703–710. https://doi.org/10.1021/acs.analchem.5b02729.
Ranchon, I., Gorrand, J.M., Cluzel, J., Droy-Lefaix, M.T., Doly, M., 1999. Functional
protection of photoreceptors from light-induced damage by dimethylthiourea and
Ginkgo biloba extract. Invest. Ophthalmol. Vis. Sci. 40, 1191–1199.
Sasaki, K., Hatta, S., 1999. Effects of bilobalide on gamma-aminobutyric acid levels and
glutamic acid decarboxylase in mouse brain. Eur. J. Pharmacol. 367, 165–173.
https://doi.org/10.1016/s0014-2999(98)00968-6.
Schmidt, B., Jaroszewski, J.W., Bro, R., Witt, M., Stærk, D., 2008. Combining PARAFAC
analysis of HPLC-PDA profiles and structural characterization using HPLC-PDA-SPE-
NMR-MS experiments: commercial preparations of St. John’s Wort. Anal. Chem. 80,
1978–1987. https://doi.org/10.1021/ac702064p.
Sprogøe, K., Stærk, D., Ziegler, H.L., Jensen, T.H., Holm-Møller, S.B., Jaroszewski, J.W.J.,
2008. Combining HPLC-PDA-MS-SPE-NMR with circular dichroism for complete
natural product characterization in crude extracts: levorotatory gossypol in
Thespesia danis. J. Nat. Prod. 71, 516–519. https://doi.org/10.1021/np800010r.
J. Zhang et al.
Stanislav, J., Shahid, M., Nakanishi, K., 2004. Isolation of ginkgolides A, B, C, J and
bilobalide from G. biloba extracts. Phytochemistry 65, 2897–2902. https://doi.org/
10.1016/j.phytochem.2004.08.026.
Sun, J., Baker, A., Chen, P., 2011. Profiling the indole alkaloids in yohimbe bark with
ultra-performance liquid chromatography coupled with ion mobility quadrupole
time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 25, 2591–2602.
https://doi.org/10.1002/rcm.5158.
Wongsomboon, P., Rattanajak, R., Kamchonwongpaisan, S., Pyne, S.G., Limtharakul, T.,
2021. Unique polyacetylenic ester-neolignan derivatives from Mitrephora tomentosa
and their antimalarial activities. Phytochemistry 183, 112615. https://doi.org/
10.1016/j.phytochem.2020.112615.
Yang, W., Ye, M., Qiao, X., Liu, C., Miao, W., Bo, T., Tao, H., Guo, D., 2012. A strategy for
efficient discovery of new natural compounds by integrating orthogonal column
chromatography and liquid chromatography/mass spectrometry analysis: its
application in Panax ginseng, Panax quinquefolium and Panax notoginseng to
characterize 437 potential new ginsenosides. Anal. Chim. Acta 739, 56–66. https://
doi.org/10.1016/j.aca.2012.06.017.
12
Phytochemistry 203 (2022) 113355
Yao, C.L., Yang, W.Z., Si, W., Shen, Y., Zhang, N.X., Chen, H.L., Pan, H.Q., Yang, M.,
Wu, W.Y., Guo, D.A., 2017. An enhanced targeted identification strategy for the
selective identification of flavonoid O-glycosides from Carthamus tinctorius by
integrating offline two-dimensional liquid chromatography/linear ion-trap-Orbitrap
mass spectrometry, high-resolution diagnostic product ions/neutral loss filtering and
liquid chromatography-solid phase extraction-nuclear magnetic resonance.
J. Chromatogr. A 1491, 87–97. https://doi.org/10.1016/j.chroma.2017.02.041.
Yao, C.L., Pan, H.Q., Wang, H., Yao, S., Yang, W.Z., Hou, J.J., Jin, Q.H., Wu, W.Y.,
Guo, D.A., 2018. Global profiling combined with predicted metabolites screening for
discovery of natural compounds: characterization of ginsenosides in the leaves of
Panax notoginseng as a case study. J. Chromatogr. A 1538, 34–44. https://doi.org/
10.1016/j.chroma.2018.01.040.
Ying, Y., Guan, Y., Shi, S.Y., Gao, Y., Zhang, R.Y., Shang, H.H., Wu, L.N., Si, D.Y., 2020.
Clinical pharmacokinetics of ginkgolides dropping pills in healthy subjects. Chin.
Tradit. Herb. Drugs 9, 2472–2479.